Microfluidic detection device having reduced dispersion and method for making same

ABSTRACT

A microfluidic detection device provides reduced dispersion of axial concentration gradients in a flowing sample. The microfluidic detection device includes a cell body and a flow path through the cell body. The flow path has an inlet segment, an outlet segment, and a central segment, which forms a detection cell. The central segment is located between and at an angle with both the inlet segment and the outlet segment. The central segment has a first junction with the inlet segment and a second junction with the outlet segment. The cell body contains two arms that can transmit light to and from the detection cell. At least a portion of a first arm is located in the first junction and at least a portion of a second arm is located in the second junction. The portions of the arms located in the junctions are situated so that fluid entering or exiting the central segment of the flow path flows around the outer surface of one of the portions. By ensuring that the flow velocity is high near the walls both at the beginning and at the end of the conduit, the configuration serves to counteract dispersion caused by the normal parabolic velocity profile of flow through a cylindrical conduit, where the fluid velocity is highest at the center. In addition, the configuration promotes efficient sweeping of the entire volume between the two arms. A method for manufacturing the microfluidic detection device is also provided.

BACKGROUND

High performance liquid chromatography (HPLC) is a technique that hasbeen used for many years as a means of separating, identifying,purifying and quantifying components of often-complex mixtures. HPLC isan important tool used by biotechnological, biomedical, and biochemicalresearch as well as in the pharmaceutical, cosmetics, energy, food, andenvironmental industries.

Conventional HPLC typically is performed using chromatographic columnswith inside diameters (I.D.'s) in the range of 2.0–4.6 mm, 4.6 mmcolumns being a common standard. However, microcolumn LC, which is themost widely accepted term to describe liquid chromatography using packedcolumns having inside diameters of 2.0 mm or less, is gaining inpopularity. Advantages of microcolumn LC include the ability to analyzesmaller sample volumes, reduction of solvent usage, and enhanced masssensitivity.

A relevant scaling factor in the design of LC systems is the square ofthe ratio of the inner diameters of the columns. The flow rate, theinjection volume and the detection volume, among other parameters, allneed to be decreased in proportion to this factor when the column I.D.is reduced in order to maintain chromatographic resolution.

Optical detectors are often used in HPLC systems to detect separatedanalytes within a fluid mixture following elution through achromatographic column. Consistent with the scaling factor mentionedabove, the volumes of optical detectors developed for use inconventional HPLC systems are too large to yield useful data when usedin microcolumn LC systems. Such detectors would fail to adequatelyresolve components with small separations.

Another quantity that must be reduced to preserve performance when thecolumn I.D. is reduced is the overall dispersion caused by thechromatographic system. Dispersion results in band broadening and theconcomitant loss of chromatographic resolution. Variance is the secondmoment of the distribution function of a chromatographic peak and is thestatistical quantity used to quantify dispersion. In the case of normaldistributions, variance is the square of the standard deviation. Thetotal variance of a peak is the sum of the variances resulting from bothcolumnar and extra-columnar, or instrumental (injector, transfer lines,detector, etc.) sources. To preserve the separation resolution achievedby the column as the column I.D. is reduced, the extra-column variancemust decrease by the ratio of the column inner diameters raised to thefourth power. While the variance contribution from instrumental sourcesis constant during a chromatographic run under the same experimentalconditions, the variance due to the column is proportional to the squareof the elution time. For this reason, the contribution of instrumentalvariance to total variance will be largest at early elution times. Anacceptable amount of variance due to instrumental dispersion isconsidered to be about 10% of the column variance at the elution time ofa non-retained peak (k=(t−t_(nr))/t_(nr)=0, where k is the retentionfactor, t is the retention time of a given peak, and t_(nr) is theretention time of a non-retained peak, which is the time it takes themobile phase to flow from the injector, through the column, to thedetector).

An example of the dramatic reduction in instrumental variance requiredto preserve the column efficiency in microscale HPLC systems can bederived by adapting information given in J. P. C. Vissers, “RecentDevelopments in Microcolumn Liquid Chromatography” J. Chromatogr. A 856(1999) 117–143. Extrapolating from Vissers, the maximum acceptablevariance due to instrumental dispersion (10% of the column variance) atk=0 for a column having a 1.0 mm inner diameter and a 15.0 cm length is90,600 nl², whereas for a column having a 300 μm inner diameter, themaximum acceptable variance due to instrumental dispersion is about 740nl² for equivalent experimental conditions.

Techniques known in the art, such as the “stacking” of analytes at thehead of a column prior to gradient elution, can be used to minimize thecontribution of instrumental variance from components prior to thecolumn. However, contribution from instrumental sources following thecolumn, notably the detection cell, cannot be reduced in this manner.Accordingly, there is a need in the art for a microfluidic detectiondevice having reduced dispersion that can perform photometricmeasurements on a flowing liquid sample and which is suitable for use inmicrocolumn LC systems.

SUMMARY

The present invention provides a microfluidic detection device thatsatisfies this need. A microfluidic detection device having features ofthe invention comprises a cell body and a flow path through the cellbody. The flow path comprises an inlet segment having a firstlongitudinal axis, an outlet segment having a second longitudinal axis,and a central segment, which forms a detection cell. The central segmenthas a third longitudinal axis and is located between and at an anglewith both the inlet segment and the outlet segment. The central segmenthas a first junction with the inlet segment and a second junction withthe outlet segment. The third longitudinal axis is transverse to boththe first longitudinal axis and the second longitudinal axis. The cellbody contains two arms that can transmit light to and from the detectioncell. At least a portion of a first arm is located in the firstjunction. At least a portion of a second arm is located in the secondjunction. The portions of the arms located in the junctions are situatedso that fluid entering or exiting the central segment of the flow pathflows around the outer surfaces. By ensuring that the flow velocity ishigh near the walls both at the beginning and at the end of the conduit,the configuration serves to counteract the dispersion caused by thenormal parabolic velocity profile of flow through a cylindrical conduit,where the fluid velocity is highest at the center. In addition, theconfiguration ensures that the entire volume between the two arms isswept efficiently, and that no “corners” or other significant volumeshaving a low flow velocity profile exist. Such regions can increasedispersion significantly since an analyte can radially diffuse intothese regions, fall behind the bulk of the material forming thechromatographic peak, and then subsequently diffuse out, resulting in a“tail” on the peak.

The arms can be substantially cylindrical. The arms can be comprised ofoptical fibers. The central segment can be substantially perpendicularto both the inlet segment and the outlet segment. Both the inlet segmentand the outlet segment can be comprised of capillaries having an innerdiameter smaller than the inner diameter of the central segment.

A method embodying features of the present invention for making amicrofluidic device comprises the steps of: coating a first surface of afirst and a second fused silica wafer with a first layer of silicon;transferring a first microconduit pattern into the first silicon layeron each wafer; transferring the first microconduit pattern from thefirst silicon layer to the first surface of each wafer so that the firstsurface of each silica wafer has the first pattern; removing the firstlayer of silicon from each wafer; securing together the first surfacesof each wafer so that the patterns on the first surface form a flowpath; inserting a portion of inserting a first optical fiber into theflow path; adhering the first optical fiber to the wafer, wherein afirst annular region exists between an inner surface of the flow pathand the portion of the first optical fiber; inserting a portion of asecond optical fiber into the flow path; and adhering the second opticalfiber to the wafers, wherein a second annular region exists between theinner surface of the flow path and the portion of the second opticalfiber, wherein light exiting one optical fiber can travel through aportion of the flow path before entering the other optical fiber. Themethod can also comprise the steps of: filling the conduits with wax;dicing the bonded pair of wafers into multiple microfluidic devices; andremoving the wax. The method can also comprise the step of inserting andadhering optical fibers and capillaries into conduits.

Thus, the present invention provides a microfluidic detection devicehaving reduced dispersion that can perform photometric measurements of aflowing liquid sample and is suitable for use in microcolumn LC systems.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawings where:

FIG. 1 is a cross-sectional view of one embodiment of a microfluidicdetection device in accordance with the present invention;

FIG. 2 is a detailed cross-sectional view of area 2 of the device ofFIG. 1; and

FIG. 3 is a cross-sectional view of the microfluidic detection device ofFIG. 1 taken on line 3—3 in FIG. 1.

DESCRIPTION

The present invention is directed to a microfluidic detection devicehaving reduced dispersion that can perform photometric measurements of aflowing liquid sample and is suitable for use in microcolumn LC systems.A microfluidic detection device according to the present invention has adetection cell that is configured so that the fluid velocity is highestnear the walls of the detection cell at the entrance and exit of thecell. This design at least partially counteracts the dispersion causedby the usual parabolic velocity profile of fluid flow through acylindrical conduit, where the fluid velocity is highest at the center.

A microfluidic detection device 10 according to the present invention isshown in FIG. 1 and includes a cell body 12 and a flow path 14 throughthe cell body. The flow path 14 has an inlet segment 16 having a firstlongitudinal axis, an outlet segment 18 having a second longitudinal asecond longitudinal axis, and a central segment 20 having a thirdlongitudinal axis and containing a detection cell 21 formed by the innersurface 38 of the central segment and the end faces of arms 28 and 32.The central segment 20 is located between both the inlet segment 16 andthe outlet segment 18 and is in fluid communication with both the inletsegment and the outlet segment. The third longitudinal axis istransverse to both the first longitudinal axis and the secondlongitudinal axis, wherein “transverse” means at an angle other than180°.

The central segment 20 has a first junction 22 with the inlet segment 16and a second junction 24 with the outlet segment 18. At least a portion26 of a first arm 28 is located in the first junction 22 and at least aportion 30 of a second arm 32 is located in the second junction 24. Ascan be seen best in FIGS. 2 and 3, the portions 26 and 30 of the arms 28and 32 located in the junctions 22 and 24 are situated so that fluidentering or exiting the central segment 20 of the flow path 14 flowsaround the outer surface 34 of one of the portions 26 and 30,respectively. This causes the velocity of the fluid flowing through thecentral segment 20 to be more uniform than if the portions 26 and 30 ofthe arms 28 and 32 were not in the junctions 22 and 24. Hence,dispersion is reduced.

The arms 28 and 32 preferably are substantially cylindrical and have asubstantially circular cross-section so that there are thin annularregions 36 between the outer surface 34 of the portions 26 and 30 andthe inner surface 38 of the central segment 20. These annular regions 36form annular openings 40 to the central segment 20. Fluid can flow inand out of the central segment 20, through the annular openings 40. Theannular openings 40 ensure that the velocity of fluid flowing throughthe central segment 20 has a maximum near the walls both near theentrance and the exit of the central segment 20. Hence, dispersion isreduced.

It is desirable that the central segment 20 be configured so that thereare no areas that are not swept by fluid, minimizing differences in flowvelocities and, hence, dispersion. Preferably, except for the annularregions, the flow path 14 is substantially tubular, i.e. has asubstantially circular cross-section, as the flow velocity profile ismore uniform in a tubular flowpath as opposed to a flow path having arectangular cross-section. In addition, the central segment 20 and theinlet segment 16 and outlet segment 18 are preferably formed in a “Z”configuration (rather than a “U” configuration) as shown in FIG. 1. In a“U” configuration (not shown), a flow streamline located near the cellwall adjacent to the inlet segment will have a shorter path to theoutlet segment than will a streamline located near the opposite cellwall. The “Z” configuration will minimize dispersion caused by flowthrough the cell by subjecting the flow streams to equal but oppositepathlength differences upon exiting the central segment. This will helppreserve the resolution of chromatographic peaks past the detectioncell, in case they are routed elsewhere following optical detection.

In some embodiments including a bend in the flow path 14 prior to thefirst junction 22 can reduce dispersion further. This bend preferably isat an angle opposite (180° different than) that of the angle formed bythe first and third longitudinal axis. For example, if the angle betweenthe first and third longitudinal axis is −90°, the bend preferably formsan angle of approximately 90° in the flow path.

The portions 26 and 30 of the arms 28 and 32 in the junctions 22 and 24can be end portions. The arms 28 and 32 can be comprised of any materialthat is capable of transmitting light of the wavelength used to analyzethe fluid passing through the detection cell 21. In the preferredembodiment, each arm 28 and 32 is comprised of an optical fiber.

The angles formed by the intersection of the central segment 20 with theinlet segment 16 and the outlet segment 18 can be any angles betweenapproximately 10 degrees and 170 degrees and need not be the same.However, if the fluid will be further analyzed after passing through thedetection cell 21, it is preferable that the angles be substantiallyequal to each other so that dispersion resulting from passage throughthe flow cell is reduced.

The inner diameters of the inlet, outlet, and central segments 16, 18,and 20, respectively, need not be the same. In some embodiments, it ispreferable that the inner diameter of the central segment 20 is largerthan the inner diameters of the inlet and outlet segments 16 and 18,respectively, so that the inner diameter of the central segment islarger than an optical fiber, while the inlet and outlet segments 16 and18, respectively, are comprised of standard, commercially availablecapillaries 42 that allow for easy connection to other microfluidicdevices. A further reason that the inlet and outlet segments 16 and 18,respectively, have a smaller inner diameter than the central segment 20in practice is that the inlet and outlet segments are initially etchedto the same depth as the central segment and capillaries are gluedwithin the inlet and outlet segments, effectively reducing theirdiameter. Therefore the effective diameter of the inlet and outletsegments when capillaries are used to connect the detection cell toother components within an analytical system are defined by thecapillary inner diameter, rather than the etch depth of the conduit thatthey are adhered to. Further, the smaller inner diameter of the inletand outlet segments 16 and 18, respectively, reduces the volume of themicrofluidic detection device 10 while not reducing the cross-section ofthe detection cell 21. Reduction of the overall volume of themicrofluidic detection device 10 minimizes dispersion.

It is important to note that flow through the annular region actuallycauses increased dispersion over that for a cylindrical tube having thesame cross-sectional area. Therefore, even though a 2-um annulus arounda ˜110-um diameter fiber has approximately the same cross-sectional areaas the 30 um diameter capillary, it has more dispersion. For thisreason, it is preferable not to employ a longer annular region thannecessary. Not using a longer annular region than necessary also helpsto keep the backpressure of fluid flowing into the central segment at alower level. It is preferable that the annular spacing, which is thespace between the inner surface of the central segment and the arm, beno larger than necessary so that as much light as possible reaches thedetector.

Annular openings in conjunction with a Teflon AF liquid waveguide flowcell are presented in U.S. Pat. No. 6,542,231. However, the patentteaches the use of a manifold or annular space prior to entering thewaveguide in order to maintain laminar flow and avoid turbulence. Whileturbulence may be an issue in conventional HPLC systems, undermicrocolumn LC conditions the Reynolds number of the fluid neverapproaches the turbulent regime and therefore the flow is alwayslaminar. The patent also gives no consideration to the optimumgeometries (principally annular spacing and length) specified as part ofthe present invention to minimize dispersion for microcolumn LCapplications, which are very sensitive to dispersion. For example, asmentioned above, flow through an annulus is actually more dispersivethan through a conduit having an equivalent, circular, cross sectionalarea. Therefore, even if an annular region is incorporated into theirflow cell design, there is no guarantee that dispersion will beminimized. In fact, it may be increased. In addition, possible methodsof fabrication of the Teflon AF liquid waveguide flow cell described inthe patent are not suited to achieving the close tolerances on verysmall dimensions required for the optimal performance of the presentinvention.

The best results are expected where the length of the annular region isapproximately 1–40 times, more preferably approximately 4–10 times, thediameter of the arms or the optical fibers as measured from the from thecenterline of either the inlet segment 16 or the outlet segment 18 tothe face of the corresponding arm and the annular spacing isapproximately 0.001 to 0.2 times, more preferably approximately0.01–0.05 times, the diameter of the arms or the optical fibers. Theannular spacing is the size of the annular region 36, which is thedistance between the outer surface 34 of one of the arms 28 and 32 andthe inner surface 38 of the central segment 20.

The cell body 12 can be comprised of a substrate such as silicon,silica, quartz, glass, or other ceramics. The cell body 12 can also becomprised of plastic or any other cell body material. The cell body 12can be constructed using laser machining, embossing, molding, casting,micromachining methods, or any other construction method known in theart. Preferably, the cell body 12 is substantially transparent tocertain wavelengths of light so that fluorescence, degenerate four wavemixing, Raman, refractive index, surface plasmon resonance, or othermeasurements can be taken through the cell body.

The detection cell volume used in microcolumn LC systems preferably isless than that of conventional systems in order to accurately analyzethe smaller sample exiting the microcolumn. The factor by whichconventional components preferably are reduced is equal to the ratio ofthe squares of the inner diameters of the chromatographic columns. Thismeans that if one wants to use 300 μm I.D. columns rather that theconventional 4.6 mm I.D. columns, the volume of the detection cell 21 ispreferably reduced by a factor of approximately 235.

However, to preserve the sensitivity of the detection cell, thepathlength over which the sample interacts with the light used indetection preferably remains close to the 1.0 cm pathlength of aconventional detection cell. Hence, the geometry of the detection cell21 is preferably optimized to have cross sectional area large enough toallow sufficient light to propagate through the detection cell 21, butsmall enough to ensure that the pathlength can be maximized while theoverall volume remains under the desired maximum value.

Light input to, and propagation through, this small detection volume canbe accomplished, for example, by using an arm 28 or 32 comprised of anoptical fiber to deliver light from a light source to one end of thedetection cell 21. The light source can be a deuterium lamp, forexample. The output of the deuterium lamp can be optimized and focusedonto the end of one of the arms 28 or 32 using a lens system orellipsoidal reflector, as just two examples of well-establishedtechniques known in the art. Any technique can be used.

A light detector, such as a CCD array-based spectrometer, is used todetect light that passes through the flowing fluid sample and travelsdown the other arm. Any light source and detector known in the art andsuitable for use with a microfluidic detection cell, as judged by one ofordinary skill in the art, can be used, including so-called singlewavelength light sources and detectors, which actually supply or detecta narrow range of wavelengths.

In this way, the detection cell 21 can be constructed having both therequired low volume and sufficient light throughput and pathlength.Since the detection device 10 is very small, the detection cell 21 canbe located very close to other components in the system, achromatographic column, for example, eliminating the need for longinterconnecting conduits, which contribute to dispersion.

The microfluidic detection device 10 can be easily incorporated as acomponent within microfabricated, chip-based, full chromatographicseparation systems in a compact and efficient manner.

Preferably, the microfluidic detection device 10 is manufactured usingthe steps detailed below, although any method known in the art can alsobe used, such as that described in A. Grosse, M. Grewe and H.Fouckhardt, “Deep Wet Etching of Fused Silica Glass for Hollow CapillaryOptical Leaky Waveguides in Microfluidic Devices,” J. Micromech.Microeng. 11, 257 (2001) and that described in U.S. patent Ser. No.10/198,223 entitled Laminated Flow Device, invented by David W. Neyer,Phillip H. Paul and Jason E. Rehm, both of which are incorporated hereinby reference for any and all purposes.

A pair of wafers are cleaned unless already clean. Standard wafer sizescan be used, 0.5–1 mm thickness, 100 mm diameter, as well as any desiredsize. The wafer can be made of silicon, glass, silica, quartz, or otherceramic materials. Further, when using silica, glass or quartz wafers, afirst surface of the pair of wafers is coated with a first layer ofsilicon. The layer can have a thickness of 1000–3000 Angstroms, forexample. The layer can be applied via low-pressure chemical vapor(LPCVD) deposition as is well known in the art. Amorphous silicon filmsare preferred related to other choices like photoresist, chrome,chrome/gold or titanium/platinum combinations for their reliability indefining channels in a fused silica substrate without edge defects thatresult from etchant-induced adhesion failure or pinholes in the film.

A first pattern for micro-conduits is transferred into the first layerof silicon on both silica wafers. The pattern can be transferred usingstandard lithography methods, like the one described in the followingparagraph.

A lithography mask can be generated from a drawing of the desiredmicro-conduit pattern, typically by a commercial vendor using a chromefilm (˜1000 Angstrom thick) on a glass substrate. If one mask is used,the same mask can be used for both wafers in the pair. Preferably, asingle mask can be used that contains a mirror plane of symmetry forthose micro-conduits that that are desired to be approximately circularin cross-section. The micro-conduit pattern preferably is designed suchthat mirror-image alignment of the pattern on each wafer will containmicro-conduit traces that substantially overlap in regions of thefluidic manifold where cylindrical channels are desired. If two masksare used, one is used for each wafer in the pair. A thin film, 1–7micrometers, for example, of photoresist (photosensitive polymer) isplaced over the layer of amorphous silicon on the pair of silica wafers.The side of each silica wafer having the thin film of photoresist isplaced proximal to or in contact with the mask. The desired microconduitpattern is transferred from the masks to the layers of photoresist byexposing the photoresist to UV light through the mask followed byappropriate development and curing of the photoresist. The microconduitpattern can be transferred from the photoresist to the silicon layer oneach wafer by etching the exposed amorphous silicon with wet chemicaletching, using a mixture of hydrofluoric, nitric, and acetic acid, forexample, or dry chemical etching, using reactive ion etching with alow-pressure (˜15-mTorr) plasma of a mixture of gases that includes SF₆,C₂ClF₅ and Ar, for example, or any other method known in the art.

After the first microconduit pattern is transferred into the first layerof silicon on both wafers, the first microconduit pattern is transferredinto the first surface of the silica wafers so that each silica waferhas a patterned surface of conduits having a substantially semi-circularcross-section. This can be accomplished by wet chemical etching of theexposed regions of the silica. The wet chemical etching can beaccomplished by timed submersion in a 49% solution of HF. Etch rates aretypically on the order of 1.3 micrometers per minute for silica. As thisetching process is isotropic, the microconduits that are formed in thewafers have a substantially semi-circular cross-section.

The photoresist can be removed using a mixture of sulfuric acid andhydrogen peroxide, for example. The first layer of silicon can beremoved by dry or wet chemical etching, as described above.

Depending on the exact design, multiple etches can be used in thefabrication of the microfluidic detection device. For example, a firstetch can be a shallow etch of about 1.5 microns and a second etch can bea deep etch of about 56 microns. Thus, the process is repeated using asecond mask.

The shallow etch can be used to define the alignment marks on the wafersand any shallow structures that are to be incorporated into the design.The alignment marks are preferably shallow etched to provide improvedalignment accuracy. In addition, the shallow etches can be used toprovide regions of slightly larger diameter, i.e. 3 microns, when theregions that are shallow etched are subsequently deep etched.

The deep-etched regions are preferably etched approximately ½ thediameter of the capillaries and optical fibers to be inserted plus about1–2 micrometers to allow a minimal space for adhesive between thecapillaries and optical fibers and the walls of the microconduit. Forexample, semicircular conduits having a radius of 56 micrometers areetched to make conduits having a circular cross-section with a 112micrometer diameter to accommodate capillaries and optical fibers havingan outside diameter of 109 micrometers. Preferably, the wafers arethoroughly cleaned with acid and base cleaning solutions so thatsurfaces of the pair of wafers are hydrophilic. In addition, the waferspreferably are also megasonically cleaned so that the surfaces of thewafers are more hydrophilic.

The first surfaces of each wafer are secured together so that thepatterns on the first surfaces form the flow path 14. The cleaned,patterned surfaces of the pair of silica wafers are substantiallyaligned and brought into contact so that the patterned surfaces formconduits having a substantially circular cross-section. The conduits canform the flow path 14 and a place to insert the arms 28 and 32 and,optionally other microfluidic components. Preferably, the alignment isaccurate to within 3 micrometers. The patterned surfaces can be alignedusing a commercially available wafer alignment device, such as theElectronic Visions EV520 aligner, which allows visual alignment of thetwo wafers while they are maintained co-planar with a very smallseparation by placing removable thin (40 microns) spacers between thewafers and avoiding contact of the two wafers prior to completealignment through the adjustment of high precision positioning stages.With the alignment complete, the wafers are clamped with the spacersremaining between the wafers. A modest pressure (approximately 2–20 psi)is applied at the center of the wafers, normal to the plane of thewafers. At this point, a weak attachment between the wafers occurs asindicated by the visually observable bonding front that moves from thecenter to the edge of the wafer. As the bonding front forms, the spacersare removed so that the entire wafer finishes bonding.

The pair of wafers is heated so that they bond together permanently.Heating the wafers (to approximately 1165° C. for silica wafers) forabout 4–8 hours is sufficient to drive a dehydration reaction at theinterface of the two wafers resulting in an interfacial bonding of thetwo wafers. The exact bonding temperature is dependant on the materialsof construction of the wafer. The result is a strong wafer bond in whichthe interface essentially disappears and the resultant part is a solidcomponent in which microconduits of substantially circular cross sectionexist for the introduction of fluid, capillaries, optical fibers,electrical leads, etc.

After bonding, the conduits can be filled with wax or some othersuitable sacrificial material to avoid particulate contamination of themicroconduits when the wafers are diced into multiple microfluidicdevices 10. A diamond saw can be used to dice the wafers. Removal of thewax can be accomplished by pyrolysis of the wax. 650° is a sufficienttemperature for pyrolysis. Since the microfluidic devices can be verysmall, dicing a single pair of bonded silica wafers can yield a largenumber of microfluidic detection devices 10 and hence, the cost ofmanufacture of the devices can be lessened.

At this point, the resulting microfluidic devices 10 are ready forinsertion and adhesion of fiber optic and fluidic-capillary components.The portions 26 and 30 of the arms 28 and 32 are inserted into the flowpath 14. The arms are then adhered to the wafer so that annular regions36 exist between the inner surface 38 of the central segment 20 and theportions 26 and 30 of the arms 28 and 32. The arms 28 and 32 arepositioned so that light exiting the face of one arm can enter the faceof the other arm after traveling through a portion of the flow path 14.

In the preferred embodiment of the microfluidic detection device 10, thebonded wafers form the cell body 12. A first arm 28, preferably anoptical fiber, is inserted into a conduit that forms the central segment20 so that at least a portion 26 of the first arm is located in thefirst junction 22. A second arm 32, preferably an optical fiber, isinserted into the central segment 20 so that at least a portion 30 ofthe second arm 32 is located in the second junction 24. Annular regions36 exist between the inner surface 38 of the central segment 20 and theportions 26 and 30 of the arms 28 and 32. Capillaries 42 can be insertedinto conduits in the cell body 12 to form the inlet and outlet segments16 and 18, respectively. The capillaries 42 are not necessary althoughthey are preferred for two reasons: 1) capillaries allow themicrofluidic detection device to easily connect to other microfluidicdevices and 2) the insertion of the capillaries makes the inner diameterof the inlet and outlet sections 16 and 18, respectively, smaller andthe smaller inner diameter reduces the volume of the microfluidicdetection device 10 while not reducing the cross-section of thedetection cell 21.

The arms 28 and 32 and the capillaries 42 preferably are fixed to thecell body 12, via an adhesive 44. The adhesive 44 can be a UV activatedoptical cement or other type of glue, an inorganic seal, such as asolgel or an aerogel solution, or any other suitable adhesive known inthe art. Alternatively the capillaries 42 and arms 28 and 32 can bedirectly fused to the cell body 12 using a CO₂ laser or any otherappropriate directed heat source known in the art.

The arm and capillary connections are very robust since the area overwhich the adhesive 44 can act is large compared to the face of the arms28 and 32 or capillaries 42. This allows high pressures to be applied tothe system without causing the connections to fail. Microfluidicdetection devices 10 manufactured in the manner described above have thecapability to perform at pressures exceeding 5000 psi since the pair ofsilica wafers are directly bonded.

In operation, fluid that has just passed through a chromatographiccolumn enters the inlet segment 16. The fluid flows through the inletsegment 16, the central segment 20, and the outlet segment 18. Afterexiting the outlet segment 18, the fluid may flow to waste.Alternatively the fluid may flow to other detection modules, such as theinput to a mass spectrometer, or it may be selectively directed alongdifferent pathways using some sort of valving arrangement depending oninformation gleaned by the detector. For example, the output may bespotted onto MALDI (matrix-assisted laser desorption and ionization)plates only when analyte is confirmed to be present by the opticaldetector. As fluid flows through the central segment 20, light from alight source exits one arm 28 or 32 and passes through fluid. Much ofthe light not absorbed by the fluid enters the other arm 28 or 32, whichpreferably guides the light to a wavelength-dispersed linear arraydetector. However, depending on the length of the central segment 20,the light may have at least one occasion to reflect off of the wall.Since the index of refraction of the silica wall is higher than thefluid, conditions for total internal reflection are not satisfied.Rather, Fresnel reflection occurs, with a high probability ofreflection, especially as the angle of incidence approaches 90 degrees.Nonetheless, the light has some probability of escaping the cell eachtime it must undergo a reflection. Some of the light will alsoback-reflect at the entrance surface of the second fiber (˜2%). Andlastly, FIG. 3 illustrates that it is possible for light to miss theentrance surface of the second fiber and be lost to the detector as itpropagates down the annular region. This can be a significant fractionof the light, roughly equal to ((d₂₀)²–(d₂₈)²)/(d₂₀)² from geometricarguments, where d₂₈ is the diameter of the arms and d₂₀ is the diameterof the central segment. This is the main motivation for minimizing theannular spacing.

In an exemplary microfluidic detection device embodying the invention,semicircular conduits having a radius of approximately 57.5 μm wereetched into wafers having a 1 mm thickness. The wafers were aligned anddirectly bonded as described herein to form circular conduits having adiameter of approximately 115 μm. The arms were optical fibers. Both theoptical fibers and the conduits used had an outer diameter ofapproximately 110 μm. The inner diameter of the capillaries used in theinlet and outlet segments was about 30 μm. The optical pathlengthbetween the optical fibers was about 4 mm. The capillaries and fiberswere glued into the wafers using UV-activated optical cement. Eachoptical fiber extends into the central segment approximately 1000 μm andthe annular spacing is approximately 2–3 μm. Optimally, for an opticalfiber having a diameter of 110 μm, each optical fiber extends into thecentral segment 20 by a distance ranging from approximately 50–4000 μmfrom the centerline of either the inlet segment 16 or the outlet segment18.

Although the present invention has been described in considerable detailwith reference to certain preferred versions thereof, other versions arepossible. For example, the angles between the central segment and theinlet and outlet segments can be approximately 20°, 80°, 50°, etc.Further, the materials for the substrate, the capillaries and the armscan be different than those specified herein without significantlyaffecting the performance of the device. Therefore, the spirit and scopeof the appended claims should not be limited to the description of thepreferred versions contained herein.

All features disclosed in the specification, including the claims,abstracts, and drawings, and all the steps in any method or processdisclosed, may be combined in any combination, except combinations whereat least some of such features and/or steps are mutually exclusive. Eachfeature disclosed in the specification, including the claims, abstract,and drawings, can be replaced by alternative features serving the same,equivalent or similar purpose, unless expressly stated otherwise. Thus,unless expressly stated otherwise, each feature disclosed is one exampleonly of a generic series of equivalent or similar features.

Any element in a claim that does not explicitly state “means” forperforming a specified function or “step” for performing a specifiedfunction should not be interpreted as a “means” or “step” clause asspecified in 35 U.S.C. §112.

1. A microfluidic detection device comprising: (a) a cell body which iscomposed of a ceramic material; (b) a flow path through the cell body,the flow path comprising: (i) an inlet segment having a firstlongitudinal axis; (ii) an outlet segment having a second longitudinalaxis; and (iii) a central segment located between the inlet segment andthe outlet segment and in fluid communication with both the inletsegment and the outlet segment, the central segment having an innersurface which is exposed to fluid flowing along the flow path and whichis composed of the ceramic material; wherein the central segment has afirst junction with the inlet segment and a second junction with theoutlet segment and wherein the central segment has a third longitudinalaxis, the third longitudinal axis being transverse to the firstlongitudinal axis and the second longitudinal axis; (c) a first opticalfiber having a portion located in the first junction so that a firstsubstantially annular region is formed between the first optical fiberand the inner surface of the central segment, wherein the first opticalfiber has a diameter and the first annular region has a length that isapproximately 1–40 times the diameter of the first optical fiber; and(d) a second optical fiber having a portion located in the secondjunction so that a second substantially annular region is formed betweenthe second optical fiber and the inner surface of the central segment,wherein the second optical fiber has a diameter and the second annularregion has a length that is approximately 1–40 times the diameter of thesecond optical fiber; wherein the portions of the optical fibers locatedin the junctions are situated so that fluid entering the central segmentof the flow path flows through one of the annular regions and fluidexiting the central segment of the flow path flows through the otherannular region.
 2. The device of claim 1 wherein the length of the firstannular region is approximately 4–10 times the diameter of the firstoptical fiber.
 3. The device of claim 2 wherein the length of the secondannular region is approximately 4–10 times the diameter of the secondoptical fiber.
 4. The device of claim 1 wherein the distance between theinner surface of the central segment and the first optical fiber isapproximately 0.001–0.2 times the diameter of the first optical fiber.5. The device of claim 4 wherein the distance between the inner surfaceof the central segment and the second optical fiber is approximately0.001–0.2 times the diameter of the second optical fiber.
 6. The deviceof claim 4 wherein the distance between the inner surface of the centralsegment and the first optical fiber is approximately 0.01–0.05 times thediameter of the first optical fiber.
 7. The device of claim 6 whereinthe distance between the inner surface of the central segment and thesecond optical fiber is approximately 0.01–0.05 times the diameter ofthe second optical fiber.
 8. The device of claim 1 further comprising:(e) a light source in optical communication with the first opticalfiber; and (f) a light detector in optical communication with the secondoptical fiber.
 9. The device of claim 1 wherein each of the opticalfibers is substantially cylindrical.
 10. The device of claim 1 whereinthe third longitudinal axis is substantially perpendicular to both thefirst longitudinal axis and the second longitudinal axis.
 11. The deviceof claim 1 wherein each of the inlet segment and the outlet segment hasan inner diameter smaller than the inner diameter of the centralsegment.
 12. The device of claim 1 wherein the ceramic material at theinner surface of the central segment is an etched ceramic material. 13.The device of claim 1 wherein the ceramic material at the inner surfaceof The central segment is etched silica.
 14. The device of claim 1wherein each of the first and second optical fibers has a substantiallycircular cross-section and an outer diameter, and the central segmentcomprises a conduit having a substantially circular cross-section havingan inner diameter larger than the outer diameter of the optical fibers.15. The device of claim 1 wherein (i) the inlet segment comprises aninlet capillary tube secured within an inlet conduit, (ii) the outletsegment comprises an outlet capillary tube secured within an outletconduit, and (iii) the central segment comprises a central conduit; theinlet conduit, the outlet conduit and the central conduit havingsubstantially the same substantially circular cross-section.
 16. Thedevice of claim 1 wherein the cell body comprises first and secondceramic wafers, each of the wafers including a mating surface having apattern etched thereon, and the mating surfaces being bonded together sothat the etched patterns together provide the flow path.
 17. The deviceof claim 1 wherein (i) the inlet segment comprises an inlet capillarytube secured within an inlet conduit, (ii) the outlet segment comprisesan outlet capillary tube secured within an outlet conduit, and (iii) thecentral segment comprises a central conduit; the inlet conduit, theoutlet conduit and the central conduit having substantially the samesubstantially circular cross-section.
 18. A microfluidic detectiondevice comprising: (a) a cell body which is composed of a ceramicmaterial; (b) a flow path in the cell body, the flow path comprising:(i) an inlet segment having a first longitudinal axis; (ii) an outletsegment having a second longitudinal axis; and (iii) a central segmentlocated between the inlet segment and the outlet segment and in fluidcommunication with both the inlet segment and the outlet segment, thecentral segment having an inner surface which is exposed to fluidflowing along the flow path and which is composed of the ceramicmaterial; wherein the central segment has a first junction with theinlet segment and a second junction with the outlet segment and whereinthe central segment has a third longitudinal axis, the thirdlongitudinal axis being transverse to the first longitudinal axis andthe second longitudinal axis; (c) a first arm having a portion locatedin the first junction so that a first substantially annular region isformed between the first arm and the inner surface of the centralsegment, wherein the first arm has a diameter and the first annularregion has a length that is approximately 1–40 times the diameter of thefirst arm; and (d) a second arm having a portion located in the secondjunction so that a second substantially annular region is formed betweenthe second arm and the inner surface of the central segment, wherein thesecond arm has a diameter and the second annular region has a lengththat is approximately 1–40 times the diameter of the second arm; whereinthe portions of the arms located in the junctions are situated so thatfluid entering the central segment of the flow path flows through one ofthe annular regions and fluid exiting the central segment of the flowpath flows through the other annular region.
 19. The device of claim 18wherein (i) the length of the first annular region is approximately 4–10times the diameter of the first optical fiber; and (ii) the length ofthe second annular region is approximately 4–10 times the diameter ofthe second optical fiber; (iii) the distance between the inner surfaceof the central segment and the first optical fiber is approximately0.01–0.05 times the diameter of the first optical fiber; and (iv) thedistance between the inner surface of the central segment and the secondoptical fiber is approximately 0.01–0.05 times the diameter of thesecond optical fiber.
 20. The device of claim 18 wherein the cell bodyis transparent to wavelengths of light permitting measurements offluorescence, degenerate four wave mixing, Raman, refractive index, orsurface plasmon resonance to be taken through the cell body.
 21. Thedevice of claim 18 wherein the ceramic material at the inner surface ofthe central segment is etched silica.
 22. The device of claim 18 whereineach of the first and second arms has a substantially circularcross-section and an outer diameter, and the central segment comprises aconduit having a substantially circular cross-section having an innerdiameter larger than the outer diameter of the arms.
 23. The device ofclaim 18 wherein the cell body comprises first and second ceramicwafers, each of the wafers including a mating surface having a patternetched thereon, and the mating surfaces being bonded together so thatthe etched patterns together provide the flow path.
 24. A microfluidicdetection device comprising: (1) a cell body which is composed of aceramic material and which comprises first and second ceramic wafers,each of the wafers including a mating surface having a pattern etchedthereon, the mating surfaces being bonded together so that the patternstogether provide a flow path through the cell body, the flow pathcomprising: (i) an inlet segment having a first longitudinal axis; (ii)an outlet segment having a second longitudinal axis; and (iii) a centralsegment which is located between the inlet segment and the outletsegment and in fluid communication with both the inlet segment and theoutlet segment, the central segment having a substantially cylindricalinner surface which is exposed to fluid flowing along the flow path andwhich is composed of the ceramic material; wherein the central segmenthas a first junction with the inlet segment and a second junction withthe outlet segment and wherein the central segment has a thirdlongitudinal axis, the third longitudinal axis being transverse to thefirst longitudinal axis and the second longitudinal axis; a firstsubstantially cylindrical optical fiber having a first diameter andhaving a portion located in the first junction so that a firstsubstantially annular region is formed between the first optical fiberand the inner surface of the central segment, the first annular regionhaving a length approximately 1–40 times the first diameter; and asecond substantially cylindrical optical fiber having the first diameterand having a portion located in the second junction so that a secondsubstantially annular region is formed between the second optical fiberand the inner surface of the central segment, the second annular regionhaving a length approximately 1–40 times the first diameter; wherein theportions of the optical fibers located in the junctions are situated sothat fluid entering the central segment of the flow path flows throughone of the annular regions, and fluid exiting the central segment of theflow path flows through the other annular region.
 25. The device ofclaim 24 wherein (i) the length of the first annular region isapproximately 4–10 times the diameter of the first optical fiber; and(ii) the length of the second annular region is approximately 4–10 timesthe diameter of the second optical fiber; (iii) the distance between theinner surface of the central segment and the first optical fiber isapproximately 0.01–0.05 times the diameter of the first optical fiber;and (iv) the distance between the inner surface of the central segmentand the second optical fiber is approximately 0.01–0.05 times thediameter of the second optical fiber.
 26. The device of claim 24 whereinthe ceramic material at the inner surface of the central segment isetched silica.
 27. The device of claim 24 wherein (i) the inlet segmentcomprises an inlet capillary tube secured within an inlet conduit, (ii)the outlet segment comprises an outlet capillary tube secured within anoutlet conduit, and (iii) the central segment comprises a centralconduit; the inlet conduit, the outlet conduit and the central conduithaving substantially the same substantially circular cross-section.